Method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling

Abstract

The present invention discloses a method for extracting chondroitin sulfate by enzymatic-deep eutectic solvent coupling, comprising the steps of extracting chondroitin sulfate from animal cartilage tissue raw material using a protease-assisted deep eutectic solvent, wherein the protease comprises one or both of a serine protease and a metalloprotease, and the raw materials for preparing the deep eutectic solvent include betaine and glucose. The present invention further claims protection for a chondroitin sulfate product prepared based on the protease-assisted deep eutectic solvent method for extracting chondroitin sulfate. The present invention utilizes a protease-assisted deep eutectic solvent to extract chondroitin sulfate, greatly improving both extraction efficiency and extraction purity, and the extraction method is environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for extracting chondroitin sulfate by coupling an enzyme method with a deep eutectic solvent. Background Art

[0002] Chondroitin sulfate (CS) is a linear glycosaminoglycan (GAG) composed of repeating disaccharide units linked by alternating β-1→3 linkages to N-acetyl-d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) via β-1→4 glycosidic bonds. CS is considered a structure-modifying drug and is commonly found in connective tissue, synovial fluid, hyaline cartilage, and bone, where it exerts structural and biophysical effects. CS is widely used as an active ingredient in dietary supplements and pharmaceutical formulations for the protection and reconstruction of articular cartilage, primarily for its anti-inflammatory properties. The vast majority of CS used in dietary and pharmaceutical formulations is derived from animals, such as through extraction from cartilage tissue and organs of cattle, pigs, and poultry.

[0003] The covalent bond between GAGs and core proteins must be broken to release CS. Alkaline, enzymatic, hydrothermal, and combinations of these methods can be used to degrade proteoglycans and are currently widely used in the production of CS. Alkaline hydrolysis is a traditional and common method because it disrupts glycopeptide and hydrogen bonds within glycoproteins, increasing CS solubility and demonstrating effectiveness in industrial CS production. However, this method consumes significant amounts of water and produces alkaline wastewater, which poses a significant environmental burden. The protease method can also produce CS, but its low extraction efficiency, low product purity, and high overall cost make it less common in industrial production. Compared to the aforementioned two methods, the hydrothermal method is becoming increasingly mainstream for CS production. However, this method requires mixing the materials with water in a suitable ratio, heating them to above 120°C, and maintaining this temperature for at least 2 hours. This method is energy-intensive and requires a pressure vessel for the extraction process, placing high demands on the equipment.

[0004] Deep eutectic solvents (DESs), a class of green solvents, have been widely used in recent years for the extraction of functional components such as polysaccharides, flavonoids, and polyphenols. DESs are primarily composed of hydrogen bond donor (HBD) and acceptor (HBA) molecules in a specific molar ratio. They are liquid solvents at room temperature and exhibit strong eutectic behavior. The highly organized structure, guided by hydrogen bonding interactions, provides DESs with significant advantages, such as biodegradability, low cost, solute stability, and ease of preparation. The solubility of different compounds in NADESs is influenced by the combination of HBD and HBA, enriching the design possibilities of their composition and stimulating their application in extraction. This implies that there is a certain correspondence between the target compound and the composition of the DES, and the extraction efficiency of the target compound can be improved by selecting the appropriate components and ratios. The applicant's prior patent application, entitled "Chondroitin Sulfate Extraction Process," with publication number CN117209624A, disclosed a method for extracting chondroitin sulfate using a deep eutectic solvent. This method improved the extraction rate of chondroitin sulfate to a certain extent. However, the above method still has room for improvement in terms of CS extraction rate, product purity, energy conservation and consumption reduction, etc. Summary of the Invention

[0005] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling. By using this method, the CS extraction efficiency is greatly improved, the extraction purity is also significantly improved, and the extraction method is green and environmentally friendly.

[0007] To achieve these objects and other advantages according to the present invention, a method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling is provided, comprising the step of extracting chondroitin sulfate from a cartilage raw material using a protease-assisted deep eutectic solvent, wherein the protease comprises one or both of a serine protease and a metalloprotease, and the raw materials for preparing the deep eutectic solvent comprise betaine and glucose.

[0008] Preferably, the mass of the protease accounts for 0.5-1.0% of the mass of the cartilage raw material.

[0009] The present invention further claims a method for preparing the deep eutectic solvent comprising:

[0010] Mix betaine and glucose in a molar ratio of 1:4, add 20-60% water of the total mass of betaine and glucose, and stir thoroughly at 70-90°C until a uniform transparent liquid is formed.

[0011] Preferably, the step of extracting chondroitin sulfate from the cartilage raw material further includes the operation of preparing the cartilage raw material into bone powder, the material-liquid ratio of the low eutectic solvent to the cartilage raw material is 20~50:1mL / g, the extraction temperature is 40~80℃, and the extraction time is 2~8h.

[0012] Preferably, the step of extracting chondroitin sulfate from the cartilage raw material further includes an enzyme inactivation step, wherein the enzyme inactivation step includes placing the extraction system in a water bath at 90-100° C. for 10-20 minutes.

[0013] Preferably, the step of enzyme inactivation further includes the operation of separating chondroitin sulfate, and the step of separating chondroitin sulfate includes:

[0014] The extract after the water bath is centrifuged at a speed of 4000-6000 r / min for 20-40 minutes, filtered through multiple layers of gauze, and the supernatant is collected. 70-90% ethanol by volume is added to the obtained supernatant and placed at 4°C for 20-36 hours. The extract is centrifuged at a speed of 10000-20000 r / min for 10-20 minutes to separate the suspension and the primary precipitate. The operation of the supernatant is repeated on the suspension to obtain a secondary precipitate. The primary precipitate and the secondary precipitate are combined and placed in a 10KDa dialysis bag, dialyzed in ultrapure water, and the dialysate is collected and freeze-dried to obtain the chondroitin sulfate, wherein the volume ratio of the ethanol to the supernatant is 2-10:1.

[0015] Preferably, the method for extracting chondroitin sulfate using an enzyme-assisted deep eutectic solvent further comprises: removing ethanol from the supernatant and recovering the deep eutectic solvent.

[0016] Preferably, the cartilage raw material comprises bovine laryngeal cartilage.

[0017] The present invention further claims protection for a chondroitin sulfate product, wherein the chondroitin sulfate product comprises chondroitin sulfate, and the chondroitin sulfate is prepared by the method of extracting chondroitin sulfate with a low eutectic solvent assisted by the enzyme.

[0018] The present invention has at least the following beneficial effects: the present invention proposes for the first time a method for extracting chondroitin sulfate using a protease and a deep eutectic solvent composite system, and screens the type and dosage of the enzyme in the composite system, and the composition, ratio, and dosage of the deep eutectic solvent; the screened protease and deep eutectic solvent composite system of the present invention greatly improves the extraction rate of chondroitin sulfate; in the enzymatic-deep eutectic solvent coupled method for extracting chondroitin sulfate, the deep eutectic solvent has a high recovery rate, the extraction method is environmentally friendly, and the economic cost is low; and the enzyme-assisted deep eutectic solvent extraction of chondroitin sulfate preferentially breaks the covalent bond between GAG and core protein without destroying the polysaccharide structure of chondroitin sulfate.

[0019] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a bar graph showing the hydrolysis degree of bovine laryngeal cartilage after NADES-2 enzymatic hydrolysis under different conditions in one embodiment of the present invention; (a) reaction temperature; (b) reaction time;

[0021] Figure 2 This is a bar graph of the extraction rates of CS using different NADESs according to another embodiment of the present invention;

[0022] Figure 3 This is a graph showing the difference in CS component changes extracted by different NADESs in another embodiment of the present invention;

[0023] Figure 4 This is an infrared spectrum of CS in another embodiment of the present invention;

[0024] Figure 5 1H-NMR and 13C-NMR diagrams of CS in another embodiment of the present invention; (a) standard; (b) water-enzyme group; (c) NADES-2-enzyme group;

[0025] Figure 6 This is a graph showing the change in CS extraction rate and purity after multiple recycling of NADES-2 in another embodiment of the present invention;

[0026] Figure 7 A ball-and-stick model diagram of another embodiment of the present invention; (a) disaccharide structure of CS; (b) NADES-2; (c) NADES-2-CS complex;

[0027] Figure 8 Electrostatic surface diagram of another embodiment of the present invention; (a) disaccharide structure of CS; (b) NADES-2; (c) NADES-2-CS complex;

[0028] Figure 9 A diagram showing different molecular interactions between NADES-2-CS complex systems in another embodiment of the present invention;

[0029] Figure 10 RDG analysis diagram of the NADES-2-CS complex in another embodiment of the present invention; (a) isosurface; (b) scatter plot. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0031] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0032] The present application provides a method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling, comprising the step of extracting chondroitin sulfate from cartilage raw materials using a protease-assisted deep eutectic solvent, wherein the protease comprises one or both of a serine protease and a metalloprotease, and the raw materials for preparing the deep eutectic solvent include betaine and glucose.

[0033] In the above technical solution, when the serine protease and metalloprotease are combined to assist the deep eutectic solvent in extracting chondroitin sulfate, the serine protease and metalloprotease are mixed in any proportion.

[0034] In one embodiment, the mass of the protease accounts for 0.5-1.0% of the mass of the cartilage raw material.

[0035] In one embodiment, the preparation method of the deep eutectic solvent comprises:

[0036] Mix betaine and glucose in a molar ratio of 1:4, add 20-60% water of the total mass of betaine and glucose, and stir thoroughly at 70-90°C until a uniform transparent liquid is formed.

[0037] In one embodiment, the step of extracting chondroitin sulfate from the cartilage raw material also includes the operation of preparing the cartilage raw material into bone powder, the material-liquid ratio of the low eutectic solvent to the cartilage raw material is 20~50:1mL / g, the extraction temperature is 40~80℃, and the extraction time is 2~8h.

[0038] In one embodiment, the step of extracting chondroitin sulfate from the cartilage raw material further includes an enzyme deactivation step, wherein the enzyme deactivation step includes placing the extraction system in a water bath at 90-100° C. for 10-20 minutes.

[0039] In one embodiment, the step of enzyme inactivation further includes an operation of separating chondroitin sulfate, and the step of separating chondroitin sulfate includes:

[0040] The extract after the water bath is centrifuged at a speed of 4000-6000 r / min for 20-40 minutes, filtered through multiple layers of gauze, and the supernatant is collected. 70-90% ethanol by volume is added to the obtained supernatant and placed at 4°C for 20-36 hours. The extract is centrifuged at a speed of 10000-20000 r / min for 10-20 minutes to separate the suspension and the primary precipitate. The operation of the supernatant is repeated on the suspension to obtain a secondary precipitate. The primary precipitate and the secondary precipitate are combined and placed in a 10KDa dialysis bag, dialyzed in ultrapure water at 4°C for 2-4 days, and the dialysate is collected and freeze-dried to obtain the chondroitin sulfate, wherein the volume ratio of the ethanol to the supernatant is 2-10:1.

[0041] In one embodiment, the method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling further comprises: removing ethanol from the supernatant and recovering the deep eutectic solvent.

[0042] In one embodiment, the cartilage raw material includes bovine laryngeal cartilage. In the method for extracting chondroitin sulfate using an enzyme-assisted low eutectic solvent provided in the present application, the cartilage raw material can be selected from pig cartilage, bovine cartilage and fish cartilage. Preferably, the cartilage raw material is selected from bovine laryngeal cartilage.

[0043] The present invention further claims protection for a chondroitin sulfate product, comprising chondroitin sulfate, produced using the enzymatic-deep eutectic solvent coupled extraction method. The chondroitin sulfate product includes, but is not limited to, a bone health dietary solid beverage, Yijiekang compressed candy tablets, and a sports nutrition oral solution.

[0044] The following is a specific embodiment of the present application:

[0045] 1. Materials and Methods

[0046] 1.1 Test materials and reagents

[0047] Bovine laryngeal cartilage raw materials were purchased from Hebei Fucheng Wufeng Food Co., Ltd. from 48-month-old yellow cattle. Meat residue was removed, the bones were thoroughly washed with running water, and dried within the red line of 55°C for 48 hours before being ground in a grinder, ball-milled, sieved, and sealed for later use. Betaine, sucrose, glucose, urea, 1,4-butanediol, ethylene glycol, glycerol, ethanol, methanol, and carbazole were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (analytical grade). Concentrated sulfuric acid, concentrated hydrochloric acid, sodium sulfate, potassium sulfate, boric acid, gelatin, barium chloride, 1-phenyl-3-methyl-5-pyrazolone (PMP), chloroform, potassium bromide, and trifluoroacetic acid were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. (analytical grade). BSA standard was purchased from Beijing Solebao Technology Co., Ltd. (analytical grade). CS standard (bovine chondroitin sulfate standard) was purchased from Shandong Xinli Biological Products Co., Ltd. (analytical grade).

[0048] 1.2 Preparation method of deep eutectic solvent

[0049] The hydrogen bond donor (HBD) and the hydrogen bond acceptor (HBA) were mixed in a specified ratio, and 40 wt % of water was added, and the mixture was stirred in a magnetic stirrer at 80 °C for 1 h until a homogeneous transparent liquid was formed.

[0050] 1.3 Methods for determining the composition and structure of chondroitin sulfate

[0051] The degree of protein hydrolysis of cartilage hydrolysate was determined by the o-phthalaldehyde (OPA) method; the sulfate ion content in CS was determined by the gelatin turbidimetry method; the uronic acid content in CS was determined by the sulfuric acid-carbazole method; and the protein content in CS was determined by the BCA method. The remaining content after removing the protein content was used to indicate the purity of CS.

[0052] The structure of CS was analyzed using FT-IR. Potassium bromide was dried at 105°C until it was anhydrous. A small amount of sample was mixed with anhydrous potassium bromide and ground into a potassium bromide tablet containing 0.5% sample. The FT-IR instrument was used for scanning, with the scan parameters set to 64 times and a resolution of 4 cm. -1 , scanning range is 4000-400cm -1 to obtain the infrared spectrum of the sample.

[0053] 1H-NMR and 13C-NMR spectra of CS were recorded using an Agilent Pro Pulse spectrometer equipped with a 5 mm diameter tunable probe. 30 mg of sample was dissolved in 1.0 mL of heavy water (D2O, 99.997%), and the NMR spectra were recorded at 25°C and 500 MHz.

[0054] 1.4 Analysis method of monosaccharide composition

[0055] The monosaccharide composition of CS was determined by ion chromatography. A 10 mg sample was weighed and placed in a hydrolysis tube, and 4 mL of 4 M trifluoroacetic acid was added. Nitrogen gas was then introduced for 1 minute to expel air from the tube. The solution was then hydrolyzed at 120°C for 2 hours under sealed conditions. After cooling the hydrolysis solution to approximately 25°C, it was blown dry with nitrogen to remove the trifluoroacetic acid, and the volume was fixed to 10 mL with ultrapure water. After dilution, the sample was filtered through a 0.22 μm filter membrane and injected. Monosaccharide ion chromatograms were recorded at 30°C on a Dionex (Sunnyvale, CA, USA) ICS-3000 ion chromatography system. The column was eluted with 4 mM NaOH at a flow rate of 1 mL / min and a temperature of 30°C.

[0056] 1.5 Analysis of disaccharide components

[0057] The disaccharide content of CS was determined by high-performance liquid chromatography. A CS solution (1 mg / mL) was treated with chondroitinase ABC (1 U / mL) at 37°C for 1 hour. The enzymatic hydrolysis product was heated in a 100°C water bath for 5 minutes to terminate the enzymatic reaction. The sample was filtered through a 0.22 μm membrane filter and injected onto a Spherisorb 5-SAX column (4.6 mm × 150 mm) at 40°C. The sample was eluted with 50 mM NaCl, pH 4, for 4 minutes and then washed with 50 mM NaCl at a flow rate of 1 mL / min. -1 Elution was performed with a linear gradient of 5 M NaCl, pH 4, for 5–35 min.

[0058] 1.6 Molecular weight analysis method

[0059] The molecular weight of CS was measured by high-performance size exclusion chromatography (Agilent 1200 HPLC, Agilent, USA) with multi-angle light scattering (Dawn Heleos II, Wyatt Technology, USA) and refractive index detection (Agilent 1260, Agilent, USA) to determine the molecular weight of depolymerized CS. The sample was eluted with 0.1 M NaCl at a flow rate of 0.5 mL / min, and the dn / dc (refractive index increment) value was 0.135.

[0060] 1.7 Enzymatic-deep eutectic solvent coupled extraction method for chondroitin sulfate

[0061] Six NADES (40% water content) containing betaine as the HBA were selected to investigate their effectiveness in extracting CS from bovine laryngeal cartilage. 2 g of laryngeal bone powder (dry weight) was added to the NADES at a liquid-to-solid ratio of 30:1 mL / g. The pH of the enzymatic hydrolysis was adjusted appropriately, and 0.8% protease (a mixture of serine protease and metalloprotease at a mass ratio of 4:3) was added. The hydrolysis temperature was 60°C for 4 hours. The sample was inactivated by boiling water for 15 minutes, centrifuged at 5000 rpm for 30 minutes, and the supernatant was collected by filtration through six layers of gauze. Ethanol was added to the system to a volume fraction of 75%. The system was incubated at 4°C overnight and centrifuged at 10000 rpm for 15 minutes at 4°C to obtain crude polysaccharides. The above steps were repeated and centrifuged again. The precipitate was resuspended in ultrapure water and placed in a 10 kDa dialysis bag. This was dialyzed against ultrapure water at 4°C for 2 days, and the dialysate was collected and freeze-dried to obtain CS. Under the same conditions, CS was extracted using water as the solvent instead of NADES and served as a control group in this experiment. The freeze-dried powder was weighed, and the extraction rate of CS from bovine laryngeal cartilage was calculated using the equation:

[0062] CS extraction rate / %=Mcs / Mcartilage ╳ 100

[0063] In the above formula, Mcs and Mcartilage represent the mass of CS sample and the total mass of the corresponding dry cartilage, g.

[0064] 1.8 Recovery Methods of Deep Eutectic Solvents

[0065] Ethanol was removed from the filtrate on a rotary evaporator to recover the NADES. The recovered NADES did not require further purification and was directly used for CS extraction to assess its reusability. Samples designated Rn represent CS extracted from the NADES obtained from the nth recovery.

[0066] 1.9 Quantum Chemistry Methods for the Mechanism of Protease-Assisted Extraction of Chondroitin Sulfate with Deep Eutectic Solvents

[0067] The structures of water, Bet-Glu (betaine / glucose), and CS clusters were constructed using MOPAC and Gaussian 09, respectively. After semiempirical optimization at the PM6-DH+ level, the structure was optimized using Gaussian optimization at the M062X / 6-31g(d) level. Single-point optimization was performed at the M062X / 6-311+g(d,p) level, with BSSN corrections to obtain molecular electrostatic potential information. The M06-2X functional accurately describes systems with non-elliptic interactions. The lowest energy structure was selected using the Multiwfn program for AIM and RDG analysis.

[0068] The existence of the critical point BCP is defined as follows: 0.002 au < ρ (BCP) < 0.035 au, 0.024 au < 0.02 2ρ (BCP) < 0.139 au. The value marked in the figure is ρ (BCP-), and the hydrogen bond strength is obtained using the following formula, and the unit of the result is kcal / mol. .

[0069] 2. Results and Discussion

[0070] 2.1 Effect of enzymatic hydrolysis conditions on the hydrolysis degree of bovine laryngeal cartilage

[0071] The effects of reaction time and reaction temperature on the hydrolysis degree of bovine laryngeal cartilage were investigated through single factor experiments, and the optimal NADES-enzymatic hydrolysis conditions were determined. Figure 1 As shown in (a), at four temperatures between 40 and 100 °C, the hydrolysis degree of cartilage by protease in the presence of NADES first increased and then stabilized. At 60 °C, the hydrolysis degree increased significantly ( P <0.05), which may be due to the presence of hydrogen bonds within NADES. The enzyme is activated in NADES, which reduces the thermodynamic water activity in the reaction medium and provides a tight hydrogen bond network as a protective layer for the protease, maintaining the integrity of the native conformation of the protease at higher temperatures, thereby enhancing the enzyme activity. At 80°C, the activity of the enzyme decreases, and the degree of hydrolysis of the cartilage should decrease. However, due to the high temperature and the decrease in the viscosity of NADES at high temperatures, the dissolution of the cartilage is promoted, so the degree of hydrolysis of the cartilage can still maintain a stable level, and too high a reaction temperature may cause Maillard browning of the hydrolyzate, which is not conducive to the extraction of polysaccharides. However, in a system without NADES, the degree of hydrolysis of the cartilage shows a downward trend with increasing temperature, indicating that the activity of the protease may have been partially inhibited at 60°C, and the enzyme activity is greatly reduced after exceeding 60°C. Figure 1 As shown in (b), the degree of cartilage hydrolysis in the water-enzyme treatment group initially increased and then slowly decreased over time. After the enzymatic treatment time exceeded 4 hours, the degree of hydrolysis remained unchanged. The degree of cartilage hydrolysis in the NADES-2-enzyme treatment group increased with reaction time and was significantly higher than that in the water-enzyme treatment group. This indicates that the optimal reaction conditions for NADES-enzyme synergistic treatment are 60°C for 4 hours.

[0072] 2.2 Effect of different DES on the extraction efficiency of chondroitin sulfate

[0073] To evaluate the optimal solvent for protease-assisted NADES extraction, betaine was selected as the HBA due to its excellent protein stability. The polysaccharide extraction performance of six NADESs with different HBD compositions was evaluated, including two sugar-based NADESs (NADES-1 and NADES-2), one urea-based NADES (NADES-3), and three alcohol-based NADESs (NADES-4, NADES-5, and NADES-6). Among them, the hydrogen bond acceptors (HBAs) of NADES-1~NADES-6 are all betaine, and the hydrogen bond donors (HBDs) are sucrose, glucose, urea, 1,4-butanediol, ethylene glycol, and glycerol, respectively. The molar ratio of betaine to sucrose is 4:1, the molar ratio of betaine to glucose is 1:4, the molar ratio of betaine to urea is 1:2, the molar ratio of betaine to 1,4-butanediol is 1:2, the molar ratio of betaine to ethylene glycol is 1:2, and the molar ratio of betaine to glycerol is 1:2. Compared with traditional solvents, NADESs have a higher binding affinity with polysaccharides due to the van der Waals, hydrogen bond and electrostatic interactions between different solvent systems, which increases the solubility of CS. With the assistance of enzymatic hydrolysis, the yield of CS extracted from cartilage using water and six different NADES systems was calculated. Figure 2 As shown, the polysaccharide extraction rates obtained by the six NADES systems ranged from 17.8% to 20.3%, all of which were significantly higher than those of the water extraction group (16.5%) ( P <0.05), with NADES-2 having the highest extraction efficiency for CS. Urea-based NADESs, due to the denaturing effect of urea on proteases, may disrupt the spatial structure of the proteases, affecting their hydrolysis ability and resulting in lower CS extraction efficiency. For alcohol-based NADESs, the increase in hydroxyl groups makes it easier for the alcohol to form intramolecular hydrogen bonds with Bet rather than intermolecular hydrogen bonds, resulting in smaller cavities, reduced free volume, and increased viscosity, leading to lower extraction efficiency. NADES-4 has fewer hydroxyl groups and an asymmetric structure, which may contribute to its higher CS extraction efficiency compared to other alcohol-based NADESs. The superior extraction efficiency of sugar-based NADESs may be attributed to the greater compatibility of sugars with enzymes. Sucrose has a larger molecular structure and high viscosity, which may hinder protease penetration. CS, on the other hand, contains glucose in its monosaccharide composition. This structural similarity allows glucose molecules to more easily interact with the polysaccharide structure, forming a stable complex. In summary, NADES-2 was selected as the optimal solvent for the protease-assisted extraction of CS.

[0074] 2.3 Analysis of basic components of CS

[0075] The sulfate content, uronic acid content and purity of CS extracted by the six NADESs groups assisted by protease and the water-enzymatic hydrolysis group are as follows: Figure 3As shown. With the help of enzymatic hydrolysis, the interaction between NADES and CS is more likely to occur. The sulfate content represents the degree of sulfation of CS. The sulfate content of CS extracted by NADESs-enzymatic hydrolysis group ranges from 4.04% to 15.05%, with significant differences ( P <0.05), the CS sulfate content extracted by NADES-2 was the highest, which may be due to the multiple hydroxyl groups in the glucose molecular structure, which promote the sulfation of CS. Among them, NADES-4 and NADES-6 in the alcohol-based NADES were not much different from the water-enzymatic hydrolysis group (5.03%). It may be that the alcohol-based NADES has a relatively mild action force, and the low dissociation force makes it difficult to destroy the glycosidic bond between CS and the core protein. NADES-4, as an alcohol-based NADES, has a high sulfate content, which may be due to its neutral pH, which promotes the sulfation of CS. Similarly, the sulfate content of alkaline NADES-3 is also high; the uronic acid content represents the degree of separation of CS from protein. The uronic acid content of CS extracted by the NADESs-enzymatic hydrolysis group ranges from 9.26% to 15.51%, which is significantly different ( P <0.05), among which the uronic acid content of CS extracted by NADES-2 was higher at 14.38%, which may be due to the oxidative degradation of cartilage treated with glucosyl NADES, which exposed more carboxyl groups; purity represents the quality of CS, and the uronic acid content of CS extracted by NADESs-enzymatic hydrolysis group ranged from 81.78% to 92.52%, which was significantly different ( P <0.05), with NADES-2 extracting the highest purity of CS. It was determined that NADES-2 was the highest-quality processing solvent for enzyme-assisted extraction of CS, which was generally consistent with the extraction yield comparison results.

[0076] 2.4 FI-IR spectrum analysis of CS

[0077] Fourier transform infrared spectroscopy can help to evaluate the structure of polysaccharides. Figure 4 CS standard (SCS), water-enzyme-hydrolyzed CS (WECS) and NADES-2-enzyme-hydrolyzed CS (BGECS) were measured at 4000 cm −1 and 500 cm −1 The FT-IR spectra of SCS, WECS and BGECS were compared in the range of 100 nm and 100 nm, respectively. According to the relevant literature, the structural data of the functional groups and chemical bonds in CS were obtained. The results showed that the infrared spectra of SCS, WECS and BGECS were very similar, and most of the functional groups of the two were basically the same: at 3450 cm −1 There is a strong broad peak at 2891 cm −1 There are CH stretching vibration peaks of CH3 nearby; at 1650 cm −1There are strong C=O stretching vibration peaks of CH3CO-NH nearby, indicating that both contain CH3CO-NH; 1420 cm −1 The CO stretching vibration at 1566 cm −1 The NH angle vibration of CH3CO-NH was found near 1254 cm −1 Strong S=O asymmetric stretching vibration peaks of sulfate ester groups appeared, indicating that both CSs have more sulfate substituents; 1065 cm −1 The characteristic peak of CO stretching vibration of sugar ring was found, proving that both CS have complete sugar ring structure; 925 cm −1 The characteristic peak of asymmetric stretching vibration of the pyranose ring nearby also confirms that the glucuronic acid of both is β-D-pyranose glucuronic acid. −1 The COS stretching vibration peak of the sulfate group appeared nearby, indicating that the sulfate group was bound to the C4 position of N-acetyl-galactosamine (GalNAc), which means that all three CS contained 4-sulfate chondroitin, namely CS-A. −1 A weaker sulfate ester vibration peak was also observed at , which is a typical characteristic absorption peak of 6-chondroitin sulfate. Therefore, it may contain a small amount of 6-chondroitin sulfate molecules, namely CS-C.

[0078] Compared to SCS, the peak spectra of WECS and BGECS were slightly shifted, but the characteristic peaks remained in the infrared spectrum. This shift in spectral properties is likely due to intermolecular interactions with the solvent, which caused a disturbance in the chemical bond lengths and strengths of the CS. The results indicate that the major functional groups and chemical bonds in the extracted polysaccharides were not disrupted. Because infrared spectroscopy can only provide a relatively simple functional group analysis of a substance's structure, nuclear magnetic resonance analysis of the three CSs is required to confirm their detailed structures.

[0079] 2.5 CS monosaccharide, disaccharide and molecular weight analysis

[0080] As shown in Table 1, the monosaccharide composition of SCS, WECS and BGECS is mainly composed of glucosamine (GlcN), glucuronic acid (GlcA), galactosamine (GalN) and galactose (Gal). Compared with the monosaccharide composition of SCS, the content of GlcA and GalN in WECS and BGECS is reduced, which is attributed to the high acidity and high temperature. hydrolysis condition, glycosidic bondThe acetyl group of GlcN is lost due to cleavage; the Gal content increased significantly, likely due to the tetramer (4-mer) bond between CS and Ser residues and a small amount of keratin sulfate. However, these values ​​were within normal ranges and met the quality standards for CS. Compared with WECS, BGECS had higher GlcA and GalN contents and lower Gal content, indicating that the latter was of higher quality. Gal is located in the region connecting CS to the core protein, indicating that the protease-assisted NADES treatment was more effective than the water-enzyme treatment in disrupting the CS protein junction. This is consistent with the extraction efficiency results.

[0081] The disaccharide compositions of SCS, WECS, and BGECS are shown in Table 1. The ΔDi0S, ΔDi6S, and ΔDi4S contents of BGCS were 33.74%, 11.02%, and 55.24%, respectively, while those of WECS were 35.56%, 11.52%, and 52.92%, respectively. These disaccharide compositions are similar to those of the standard CS (ΔDi0S, ΔDi6S, and ΔDi4S: 32.28%, 10.17%, and 57.55%, respectively), indicating that neither treatment disrupted the CS structure. Chondroitinase ABC produced varying percentages of unsaturated disaccharides from the three CSs. The 4S / 6S ratios for SCS, WECS, and BGECS were 1.78, 1.48, and 1.63, respectively, indicating that 4-chondroitin sulfate (CS-A) predominated in all cases, consistent with the infrared data.

[0082] The molecular weight of polysaccharides influences their bioactivity. Polysaccharides with excessively high molecular weights have poor solubility and high viscosity in aqueous solutions. Consequently, these large molecules have difficulty penetrating cell membranes, resulting in relatively weak bioavailability and bioactivity. The average molecular weight of BGECS is 46.32 ± 0.19 kDa, which is between SCS (19.23 ± 0.17) and WECS (64.74 ± 0.13), with a relatively small difference from SCS. The CS quality of BGECS is significantly superior to that of WECS. This may be due to the fact that the NADES solvent, compared to water, better separates proteins from CS, promoting enzyme dissociation and resulting in a lower molecular weight. Water, however, has a weaker interaction and fails to promote enzyme activity, resulting in incomplete separation and the production of larger molecular weight CS, which may contribute to differences in bioactivity. The CS polydispersity index (PDI, Mw / Mn) is less than 1.5, indicating that the compositions of SCS, WECS, and BGECS are relatively homogeneous.

[0083] Table 1 Analysis and average molecular weight of monosaccharides and disaccharides of CS

[0084]

[0085] 2.6 NMR spectral analysis of CS

[0086] like Figure 5 WECS and BGECS 1 H-NMR showed that there were impurity peaks in the 1.5-5.0 ppm region, which may be related to the presence of protein impurities in WECS and BGECS. According to relevant literature, the signals between 3.4 and 4.8 ppm are attributed to GlcA and GalNAc protons, and the methyl signals on GalNAc, which indicates that all three contain two or more GalNAc, that is, GalNAc has multiple sulfate substitution modes. In the spectrum, 4.60, 4.55 and 4.54 ppm are the 4-sulfate-substituted GalNAc4S H-4 signals in SCS, WCS and BGCS, respectively; 4.48, 4.49 and 4.48 ppm are the 4-sulfate-substituted GalNAc H-1 signals in the three, respectively; the peak at 4.39 ppm is the 4-sulfate-substituted GlcA H-1 signal, and its signal value is higher than the GalNAcH-4 signal at 4.10 ppm; 3.94, 3.88 and 3.88 ppm are the 4-sulfate-substituted GalNAc H-2,3 signals in the three, respectively; 3.70, 4.49 and 3.70 ppm are the signals of 4-sulfate-substituted GlcA H-4 in the three; 3.50 ppm are the signals of 4-sulfate-substituted GlcA H-3 in the three; 3.28 ppm are the signals of 4-sulfate-substituted GlcA H-2 in the three; the appearance of these signals indicates that there are monosulfated disaccharides, namely CS-A, in both CS.

[0087] like Figure 5 For SCS, WECS and BGECS 13 The positions of sulfate groups in GalN were determined by C NMR spectra in the 50-100 ppm range. The three CSs showed peaks associated with C-1 of GlcA-6SO4 and GlcA-4SO4 at 104.31, 104.31, and 104.35, and 103.79, 103.84, and 103.83 ppm, respectively. Peaks at 67.59, 67.53, and 67.55 ppm were associated with C-4 of GalN-6SO4 (CSC), and peaks at 100.92, 100.81, and 101.01, 61.08, 61.03, and 61.09, and 51.49, 51.52, and 51.63 ppm were associated with C-1, C-6, and C-2 of GalN-4SO4 (CSA), respectively. The results show that in the range of 50-110 ppm, the SCS, WECS and BGECS 13C-NMR spectra showed that the content of CSA was higher than that of CSC. Although some miscellaneous peaks appeared in the NMR spectra of SCS, WECS, and BGECS, their characteristic signals did not disappear, indicating that the water-enzyme group and NADES-2-enzyme group treatments did not destroy the structure of CS.

[0088] In summary, compared with SCS, WECS and BGECS 1 H and 13 The characteristic signals in the C-NMR spectra showed almost the same shifts, indicating that WECS and BGECS have similar structures.

[0089] 2.7 Analysis of NADES Recyclability

[0090] After the enzymatic extraction of CS by NADES-2, the extraction rate and efficiency of NADES-2 decreased slightly after 5 cycles. This may be due to the increase in the number of reuses, and some collagen in the cartilage matrix and enzyme impurities that were inactivated after the reaction remained in the solvent, increasing the burden on the solvent. The results showed that ( Figure 6 ), NADES-2 maintained a good extraction rate (about 18.12%) after being reused 5 times, which was higher than the extraction rate of the water-enzymatic hydrolysis group (16.5%), and had no significant difference compared with the extraction rate of the uncirculated solvent (R0) ( P >0.05). With the increase of recycling times, the purity of the extracted CS gradually decreased. After five reuses, the purity only decreased by 4.53%. Although the macromolecular protein impurities in the regenerated NADES weakened the hydrogen bond interaction force in the solvent, it still had a good dissolution effect, indicating that the NADES prepared in this study has good cyclic stability and recyclability in the extraction of CS with the assistance of the enzymatic hydrolysis system.

[0091] 2.8 Analysis of the dissolution behavior of CS in NADES

[0092] In order to better understand the interaction between NADES and CS, the DFT calculation method was used to study the weak interaction of NADES on CS. The structure of CS (structure a) combined with NADES-2 (structure b) was simulated using AIM to obtain structure c, which is the complex of NADES-2 and CS. The three structures were optimized at the M06-2X-D3 / 6-31G(d) level, and frequency calculations were performed to ensure that there were no imaginary frequencies in the structure, and thermodynamic corrections were obtained at the same time. The optimized structures were subjected to single-point energy calculations at the M06-2X-D3 / 6-311+G(d,p) level, and GCP correction was used to eliminate BSSE. Figure 7 The optimized geometric structures of NADES-2 and CS, and NADES-2-CS complex are shown.

[0093] Quantitative analysis of the electrostatic potential surface (ESP) is an effective tool for studying molecular interactions and predicting potential reaction sites. Blue areas indicate negative ESP, white areas indicate zero ESP, and red areas indicate positive ESP. Figure 8 As can be seen from a, the carbon ring and hydroxyl hydrogen of CS have positive electrostatic potential, while the benzene ring near the β-O-4 ether bond has negative electrostatic potential. The negatively charged carboxylate group can easily form hydrogen bonds with the positively charged hydroxyl group of CS. Figure 8 As shown in (b), NADES-2 is tightly surrounded by glucose, and the charge distribution on the surface is relatively even, with only some areas with obvious negative charges around the carboxyl groups. The hydroxyl hydrogen part of NADES-2 has a positive electrostatic potential, and the carboxylate group of betaine has a negative electrostatic potential. Hydrogen bonds are formed between the negatively charged carboxylate group and the positively charged hydroxyl group in glucose. The formation of hydrogen bonds between HBD and HBA delocalizes the charge, thereby deriving the formation of NADESs and making the atoms involved in the hydrogen bonds have lower electron density. Figure 8 c. The negative charge of CS is concentrated on the sulfate group and the carboxyl group of the co-solvent, while the quaternary ammonium is more positively charged. The benzene ring near the β-O-4 ether bond readily interacts with the hydroxyl hydrogen moiety of NADES-2. This study suggests that the primary driving force for CS dissolution is the formation of hydrogen bonds between CS and NADES-2.

[0094] The interaction energy between NADES-2 and CS is calculated in Table 2. The binding free energy of NADES-2 and CS is -64.48 kcal / mol, and the binding enthalpy is -84.53 kcal / mol. This indicates that the extraction reaction is exothermic, which is favorable for the reaction and explains the dissolution behavior of CS during NADES extraction. Overall, the addition of NADES-2 to the cartilage matrix effectively improves the solubility of CS, which is consistent with previous studies.

[0095] Table 2 Mutual energy values ​​among CS, NADES-2 and NADES-2-CS complex systems

[0096]

[0097] 2.9 Analysis of the interaction between NADES and CS

[0098] AIM analysis of intermolecular hydrogen bonds revealed a close hydrogen bonding network between NADES-2 and CS, primarily concentrated in OH...O and C-H...O hydrogen bonds (Table 3). The complex hydrogen bonding network within the complex demonstrates a strong interaction between CS and NADES-2. Results indicate that the carbon ring of CS donates protons, enhancing the solubility of CS in NADES-2. Positive values ​​of electron density and ∇2ρ(BCP) indicate electrostatic interactions, while negative values ​​indicate covalent interactions. As shown in Table 3, hydrogen bonds are formed between CS and NADES-2. The OH...O type hydrogen bonds are mainly: O104-H114...O14 type formed by the oxygen in the CS ring and the hydroxyl group on the glucose ring, and O12-H39...O159 type formed by the hydroxyl group on CS and the glucose hydroxyl group in NADES-2; C-H...O hydrogen bonds are mainly: C86-H77...O24 type formed by the sulfate group of CS and the carbon on the glucose ring in NADES-2, C133-H127...O12 type formed by the hydroxyl group of CS and the carbon on the glucose ring in NADES-2, and C54-H63...O2 type formed by the methyl group on Bet in NADES-2 and the sulfate group of CS. This application also provides an interaction diagram of the NADES-2-CS complex ( Figure 9 The carboxylic acid group of Bet is tightly surrounded by dense glucose molecules. The hydroxyl groups on the glucose ring form hydrogen bonds between the O and H atoms. Various hydrogen bonding interactions were also observed in the system, including O51-H62…O60; O100-H112…O102; O150-H161…O80; C53-H62…O135; C55-H67…O58 ( Figure 9 ), among which the O25-H46…O22 type and the O150-H161…O80 type are 0.034 Au, 0.110 Au, -6.83 kcal / mol and 0.027Au, 0.110Au, -6.17 kcal / mol, respectively, indicating that they are indeed hydrogen bonds, and the O25-H46…O22 type is much stronger than the O150-H161…O80 type.

[0099] Table 3 Hydrogen bond energy of NADES-2-CS complex

[0100]

[0101] like Figure 10 a, Reduced density gradient (RDG) analysis, whose scatter plot is based on electron density gradient, is used to detect the type and strength of non-covalent interactions as a function of real space. The redder the color, the stronger the repulsion, and the bluer the color, the stronger the attraction. The blue pie-shaped RDG isosurface indicates that strong hydrogen bonds are formed in the above-mentioned region of the NADES-2-CS complex. For comparison, Figure 10The isosurface plot of the colored RDG of the NADES-2-CS complex (b) shows that, in addition to hydrogen bonding interactions (delineated in blue), van der Waals forces (delineated in green) and steric hindrance (shown in red) also coexist between NADES and CS. Furthermore, the dense peaks for NADES-2 and CS indicate a stable interaction between the two groups. The interaction and ρ(r) values ​​near the symbol (λ2) in the middle region represent van der Waals forces, while the smaller peaks (-0.03 to -0.01) on the left represent attractive hydrogen bonds. Furthermore, the hydroxyl groups on the cyclic structure of glucose prefer to form intramolecular or intermolecular hydrogen bonds (OH⋯H). In the scatter plot of the NADES-2-CS complex, the value of the middle vertical bar is close to zero, indicating strong hydrogen bonding. The protons in NADES-2 readily dissociate, promoting solvent acidity and enhancing solubility from cartilage. CS competes with the glucose in NADES-2 for hydrogen bonding, resulting in improved CS extraction efficiency.

[0102] 3. Conclusion

[0103] Protease was added to the NADESs system to extract CS. The reaction temperature and time process conditions for the optimal enzymatic hydrolysis of CS in the NADES environment were determined based on the hydrolysis degree of the cartilage matrix. Six betaine-based NADESs were prepared using the solvent-liquid ratio and water content optimized in previous experiments. The composition of the optimal NADES in the protease-assisted NADESs extraction method was further explored based on the CS extraction rate. The results showed that the optimal enzymatic hydrolysis conditions for the protease-assisted NADES extraction method were 60°C and 4 h. Under these conditions, the CS extraction rate of cartilage treated with protease-assisted NADES-2 (betaine-glucose, molar ratio 1:4, water content 40%) was the highest, at 20.3%, which was 1.23 times that of the water-enzymatic hydrolysis method. CS composition analysis revealed that the CS (BGECS) extracted by NADES-2 was highly pure (92.52%) and bioactive (sulfate content 15.05%, uronic acid 14.38%). CS structural characterization revealed that the BGECS had a well-defined structure and low molecular weight. Recovery experiments after enzymatic hydrolysis showed that after five reuses of NADES-2, the CS extraction yield remained at 18.12%, with only a 4.53% decrease in purity. Furthermore, to explore the potential mechanism of this NADES extraction process, density functional theory (DFT) was used to calculate the binding energy of the Bet-Glu-CS complex and simulate the interaction forces within the complex. Electrostatic potential analysis indicated that the primary driving force for CS dissolution was hydrogen bonding between CS and NADES-2. The binding free energy of NADES-2 to CS was -64.48 kcal / mol, favoring the reaction. AIM analysis revealed strong hydrogen bonding between NADES-2 and CS, primarily at the OH...O and C...O hydrogen bonds. RDG analysis showed that CS competitively formed hydrogen bonds with glucose in NADES-2, resulting in improved CS extraction efficiency, indicating that protease-assisted NADES-2 can efficiently recover CS from laryngeal cartilage.

[0104] In order to verify that the addition of protease is beneficial to the improvement of chondroitin sulfate extraction rate, the following experiments were further conducted:

[0105] Example 1

[0106] Preparation of deep eutectic solvent:

[0107] Betaine and glucose were mixed in a molar ratio of 1:4, and 40% of the total mass of betaine and glucose was added into water, and the mixture was stirred thoroughly at 80°C until a uniform transparent liquid was formed.

[0108] Method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling:

[0109] 2 g of bovine laryngeal cartilage powder was added to NADES at a material-liquid ratio of 30:1, 0.8% serine protease was added, the extraction temperature was 60°C, the extraction time was 4 h, the extraction system was placed in a 100°C water bath for 15 min, the extract after the water bath was centrifuged at 5000 r / min for 30 min, and filtered with 6 layers of gauze to collect the supernatant, 75% ethanol by volume was added to the obtained supernatant and placed at 4°C for 24 h, centrifuged at 100000 r / min for 15 min to separate the suspension and the primary precipitate, the operation on the supernatant was repeated on the suspension to obtain a secondary precipitate, the primary precipitate and the secondary precipitate were combined and placed in a 10KDa dialysis bag, dialyzed in ultrapure water, the dialysate was collected and freeze-dried to obtain the chondroitin sulfate, wherein the volume ratio of the ethanol to the supernatant was 8:1.

[0110] Example 2

[0111] The preparation of deep eutectic solvent is the same as that in Example 1

[0112] In the method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling, the serine protease was replaced by a metalloprotease, and the rest was the same as in Example 1.

[0113] Example 3

[0114] The preparation of the deep eutectic solvent is the same as in Example 1.

[0115] In the method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling, the serine protease is replaced by a complex enzyme of serine protease and metalloprotease, wherein the mass ratio of serine protease to metalloprotease is 1:1, and the rest is the same as in Example 1.

[0116] Example 4

[0117] The preparation of the deep eutectic solvent is the same as in Example 1.

[0118] In the method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling, the serine protease is replaced by a complex enzyme of serine protease and metalloprotease, wherein the mass ratio of serine protease to metalloprotease is 4:3. Other steps are the same as in Example 1.

[0119] Comparative Example

[0120] The preparation of the deep eutectic solvent is the same as in Example 1.

[0121] No protease was added to the method for extracting chondroitin sulfate with a deep eutectic solvent, and the other steps were the same as in Example 1.

[0122] The extraction rates of chondroitin sulfate obtained in Examples 1 to 4 and the comparative example are shown in Table 4.

[0123] Table 4 Chondroitin sulfate extraction rates obtained from Examples 1 to 4 and Comparative Examples

[0124]

[0125] As shown in Table 4, the methods of using serine protease or metalloprotease alone as an enzyme method-deep eutectic solvent coupled extraction of chondroitin sulfate (Examples 1 and 2), or mixing serine protease and metalloprotease in any proportion as a composite enzyme method-deep eutectic solvent coupled extraction of chondroitin sulfate (Examples 3 and 4), can significantly improve the extraction rate and extraction purity of chondroitin sulfate compared to the method of extracting chondroitin sulfate using only a deep eutectic solvent (Comparative Example). This shows that serine protease and metalloprotease, whether acting alone or in combination in any proportion, have a significant synergistic effect on the extraction rate and extraction purity of chondroitin sulfate extracted by deep eutectic solvent. Among them, when serine protease and metalloprotease are mixed in a mass ratio of 4:3 (Example 4), the extraction rate and extraction purity of chondroitin sulfate assisted by deep eutectic solvent extraction are the highest. The extraction rate is increased by 113.7% compared to the extraction rate of chondroitin sulfate extracted using deep eutectic solvent alone, and the extraction purity is increased by 20.1% compared to the extraction purity of chondroitin sulfate extracted using deep eutectic solvent alone.

[0126] As described above, according to the present invention, the present invention has at least the following beneficial effects: the present invention proposes for the first time a method for extracting chondroitin sulfate using an enzyme and a deep eutectic solvent composite system, and screens the type and dosage of the enzyme in the composite system, and the composition, ratio, and dosage of the deep eutectic solvent; the screened enzyme and deep eutectic solvent composite system of the present invention greatly improves the extraction rate of chondroitin sulfate; in the enzymatic-deep eutectic solvent coupled method for extracting chondroitin sulfate, the deep eutectic solvent has a high recovery rate, the extraction method is environmentally friendly, and the economic cost is low; and the use of a protease-assisted deep eutectic solvent to extract chondroitin sulfate preferentially breaks the covalent bond between GAG and core protein without destroying the polysaccharide structure of chondroitin sulfate.

[0127] The number of equipment and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the protease-assisted deep eutectic solvent extraction method of the present invention will be readily apparent to those skilled in the art.

[0128] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and specific embodiments shown and described herein.

Claims

1. A method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling, characterized in that: The method comprises the steps of extracting chondroitin sulfate from cartilage raw materials by using a protease-assisted low eutectic solvent, wherein the protease comprises one or both of serine protease and metalloprotease, and the raw materials for preparing the low eutectic solvent comprise betaine and glucose.

2. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 1, characterized in that: The mass of the protease accounts for 0.5-1.0% of the mass of the cartilage raw material.

3. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 1, characterized in that: The preparation method of the deep eutectic solvent comprises: Mix betaine and glucose in a molar ratio of 1:4, add 20-60% water of the total mass of betaine and glucose, and stir thoroughly at 70-90°C until a uniform transparent liquid is formed.

4. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 1, characterized in that: The step of extracting chondroitin sulfate from the cartilage raw material also includes the operation of preparing the cartilage raw material into bone powder, the material-liquid ratio of the low eutectic solvent to the cartilage raw material is 20-50:1 mL / g, the extraction temperature is 40-80° C., and the extraction time is 2-8 hours.

5. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 1, characterized in that: The step of extracting chondroitin sulfate from the cartilage raw material also includes an enzyme deactivation step, which includes placing the extraction system in a water bath at 90-100° C. for 10-20 minutes.

6. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 5, characterized in that: The step of enzyme inactivation also includes the operation of separating chondroitin sulfate, and the step of separating chondroitin sulfate includes: The extract after the water bath is centrifuged at a speed of 4000-6000 r / min for 20-40 minutes, filtered through multiple layers of gauze, and the supernatant is collected. 70-90% ethanol by volume is added to the obtained supernatant and placed at 4°C for 20-36 hours. The extract is centrifuged at a speed of 10000-20000 r / min for 10-20 minutes to separate the suspension and the primary precipitate. The operation of the supernatant is repeated on the suspension to obtain a secondary precipitate. The primary precipitate and the secondary precipitate are combined and placed in a 10KDa dialysis bag, dialyzed in ultrapure water, and the dialysate is collected and freeze-dried to obtain the chondroitin sulfate, wherein the volume ratio of the ethanol to the supernatant is 2-10:

1.

7. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 6, characterized in that: Also includes: The supernatant is taken to remove ethanol and recover the deep eutectic solvent.

8. The method for extracting chondroitin sulfate by enzyme-deep eutectic solvent coupling according to claim 1, characterized in that: The cartilage raw material includes bovine laryngeal cartilage.

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